Thyroid hormone analysis by mass spectrometry

ABSTRACT

Methods, systems and kits for the simultaneous or sequential analysis of one or more hormones by mass spectrometry are disclosed. The methods require minimal sample size and minimal preparation time. The methods comprise ionizing the hormones and analyzing the hormones by mass spectrometry. In addition, methods, systems and kits for the simultaneous or sequential analysis of thyroid hormones are disclosed comprising ionization of the thyroid hormones in the negative mode using an electrospray source.

FIELD OF THE INVENTION

[0001] The present invention combines the fields of hormone analysis andmass spectrometry. In particular the invention relates to analyzingthyroid hormones using mass spectrometry.

BACKGROUND OF THE INVENTION

[0002] Hormones are biological messengers. They are synthesized byspecific tissues (glands) and are secreted into the blood. The bloodcarries them to target cells where they act to alter the activities ofthe target cells.

[0003] Hormones are chemically diverse, and are generally categorizedinto three main groups: (1) small molecules derived from amino acids,for example thyroxine, (2) polypeptides or proteins, for example insulinand thyroid-stimulating hormone, and (3) molecules derived fromcholesterol, for example steroids.

[0004] An important class of hormone is the thyroid hormones. Examplesof thyroid hormones are thyroxine (T4), free thryoxine (FT4) andtriiodothyronine (T3). T4 and T3 enter cells and bind to intracellularreceptors where they increase the metabolic capabilities of the cell byincreasing mitochondria and mitochondrial enzymes. T4 and T3 areimportant in regulating a number of biological processes, includinggrowth and development, carbohydrate metabolism, oxygen consumption,protein synthesis and fetal neurodevelopment. Synthesis of allcirculating T4 and a small percentage of circulating T3 occurs onthyroglobulin molecules located within the thyroid. The bulk of the T3present in the blood is produced enzymatically via monodeiodination ofT4 by specific intracellular deiodinases—enzymes present in thefollicular cells and the cells of target tissues [1]. In serum drawnfrom healthy human subjects, total T4 is present at about 60-fold higherconcentration than total T3. T4 acts as a prohormone, as the reservoirfor the production of T3, the active hormone. The metabolic activityassociated with thyroid hormone (TH) is initiated by T3 binding tospecific nuclear receptors within target cells. Thyroid hormoneconcentrations in blood are essential tests for the assessment ofthyroid function.

[0005] Steroids make up another important class of hormones. Examples ofsteroid hormones include estrogens, progesterone and testosterone.Estrogen is the name of a group of hormones of which there are threeprinciple forms, estrone, estradiol and estriol. Estrogens andprogesterone cause the development of the female secondary sexualcharacteristics and develop and maintain the reproductive function.Testosterone develops and maintains the male secondary sexcharacteristics, promotes growth and formation of sperm. Steroids entertarget cells and bind to intracellular receptors and then cause theproduction of mRNA coding for proteins that manifest the changes inducedby steroids.

[0006] The accurate analysis and quantfication of hormones is becomingmore important. For example, estrogen and estrogen like compounds areplaying an ever-increasing role in today's society through hormonereplacement therapy. Also, the analysis and quantification of estrogenand estrogen-like compounds helps in the management of estrogen-relateddiseases, like breast cancer. In addition, the accurate analysis andquantification of T4 and T3 is an issue recognized by those skilled inthe art. The presence of circulating iodothyronine-bindingautoantibodies that interfere with total T4 and T3 radioimmunoassays(“RIAs”) is a known phenomenon [2], [3], [4]. These autoantibodies maygive falsely high, or falsely low values of thyroid hormone measurementsdepending on the assay separation method used, and are often indiscordance with the clinical features [2], [3], [4]. Direct serum freeT4 and T3 (FT4 and FT3) measurements are a way to compensate for suchabnormal binding. However, technically, it is difficult to measure thefree hormone concentrations since these are so low. It is easier tomeasure the total (free and protein-bound) thyroid hormoneconcentrations; total hormone concentrations are measured at nanomolarlevels whereas free hormone concentrations are measured in the picomolerange and to be valid, must be free from interference by the much highertotal hormone concentrations.

[0007] Presently, the common methods of hormone analysis use immunoassaytechniques. Table 1 lists the common hormones and the current methodsfor their analysis.

[0008] For example, estriol is analyzed by a radioimmunoassay utilizingradiolabelled antigen (iodine 125) in competition with unlabelledestriol in the sample, for a known amount of antibody. The assay is readusing a gamma counter.

[0009] Androstenedione is analyzed using an enzyme immunoassaycomprising horseradish peroxidase. Unlabeled antigen in the sample is incompetition with enzyme labeled antigen for a fixed number of antibodybinding sites. The assay is read using a microtitre plate enzymeimmunoassay reader.

[0010] Several hormones are currently analyzed using a chemiluminescentimmunoassay. For example, progesterone, testosterone, cortisol and T3are analyzed using this method. The assay utilizes an assay specificantibody-coated bead. The assay is read using a photon counter.

[0011] However, the current immunoassays are disadvantageous for thefollowing reasons:

[0012] (1) Immunoassays are specific to one hormone, therefore everyhormone must be analyzed separately.

[0013] (2) Numerous kits must be purchased and procedures must belearned for each hormone being analyzed.

[0014] (3) Various instruments to read the results from the immunoassaysmust be purchased. For example, the analysis of estriol and progesteronefrom a sample requires both a gamma counter and a photon counter.

[0015] (4) The kits for the assays can be expensive.

[0016] (5) The current immunoassays lack specificity and may showapproximately 15 fold difference In results using kits from differentmanufacturers [5].

[0017] (6) The procedures involve many steps and can take a significantamount of time.

[0018] (7) In the case of a radioimmunoassay, precautions are necessarybecause of the radioisotopes involved.

[0019] Immunoassays are notoriously unreliable with more and moreliterature published supporting their lack of specificity [6-13]. Table2 shows the major differences reported by the College of AmericanPathologists program for proficiency Testing of thyroid hormones thatclearly illustrates the difference in specificity of the variousantibodies used. For example. Table 2 shows mean results betweendifferent methods reported in the College of American PathologistsProficiency Testing (CAP PT) Program can vary by a factor ofapproximately 2. Some factors such as pregnancy, estrogen therapy orgenetic abnormalities in protein binding have also reportedly madeImmunoassay methods for T4 and T3 diagnostically unreliable [2], [3],[14], [15]. Currently serum total T4 (TT4) and total serum T3 (TT3)concentrations are most commonly measured by immunoassay methods.Recently some reports of quantitative measurement of T4 and T3 by highperformance liquid chromatography (HPLC), gas chromatography massspectrometry (GC-MS), liquid chromatography mass spectrometry (LC-MS) ortandem mass spectrometry (LC-MS/MS) were published [16-20]. All thosemethods required extraction, derivatization and even priorchromatographic separation that are very time consuming [21], [22].

[0020] More recently, hormones have been analysed and quantified by massspectrometry. However, there are several disadvantages to these methods.

[0021] For example, a method of analyzing urinary testosterone anddihydrotestosterone glucuronides using electrospray tandem massspectrometry has been described [23]. The method involves a complexsystem employing high testosterone and the limit of quantification was10 ug L⁻¹ for dihydrotestosterone and (v) the method is complex.

[0022] Another publication discloses a method for the determination ofestradiol in bovine plasma by an ion trap gas chromatography-tandem massspectrometry technique [24]. The shortcomings include the following: (i)only one analyte was analyzed, (ii) 4 ml of plasma was required for theanalysis of one analyte, (iii) the limit of detection was 5 pg ml⁻¹, and(iv) derivation was required because the method employs gaschromatography.

[0023] A method for analysis of 17-hydroxyprogesterone by HPLCeleotrospray ionization tandem mass spectrometry from dried blood spotshas also been described [25]. However, this method analyses only oneanalyte at a time, and requires liquid-liquid extraction, which islaborious and time consuming, with sample extraction alone taking 50minutes to complete.

[0024] Finally, a gas chromatography mass spectrometry method to analyzethe production rates of testosterone and dihydrosterone has beendisclosed [26]. TABLE 1 METHODS AND INSTRUMENTS FOR STEROID AND THYROIDHORMONES [1]. Percentage ANALYTE of Use Instrument METHODAndrostenedione 35% DSL solid EIA 11-Deoxycortisol 50% ICN Immuchem DARIA DHEA Sulfate 39% DPC Immulite ECIA Estradiol 16% Bayer ADVIA CentaurFIA Estriol, unconjugated 25% DSL liquid RIA Estriol, Total 50% DPCCoat-a-Count RIA 17-Hydroxyprogesterone 51% DPC Coat-a-Count RIAProgesterone 23% Bayer ADVIA Centaur CIA Testosterone 29% Bayer ADVIACentaur CIA Testosterone, Free 65% DPC Coat-a-Count RIA Aldosterone 76%DPC Coat-a-Count RIA Cortisol 25% Bayer ADVIA Centaur CIA T3 29% AbbottAxsym FPIA T3, Free 31% Bayer ADVIA Centaur CIA T4 30% Abbott Axsym FPIAT4, Free 34% Abbott Axsym FPIA

[0025] TABLE 2 Problems with Immunoassays: Data acquired from CAP PTProgram 2003 Mean CAP Result for Mean CAP Result for Method GivingLowest Method Giving Analyte Value Highest Value Triiodothyronine(ng/dL) 108.5 190.2 364.8 610.1 Thyroxine (ug/dL) 5.64 10.09 1.64 3.658.73 13.12

SUMMARY OF THE INVENTION

[0026] The invention provides a fast and accurate method of hormoneanalysis and quantification using a mass spectrometer.

[0027] A plurality of hormones can be analyzed simultaneously orsequentially. The procedure allows for as little as 100 μL of a sampleto be analyzed. In addition, minimal sample preparation time isrequired.

[0028] The invention permits the analysis of hormones in a number ofcomplex matrices as they might be found in nature, e.g. the human body.For, example, hormone analysis can be performed on samples of blood,saliva, serum, plasma and urine.

[0029] There are several advantages to this invention:

[0030] (1) It provides a total and specific analysis for hormones in asample. The present method allows for the analysis of many hormonessimultaneously or sequentially.

[0031] (2) The procedure does not require an immunoprecipitationreaction. The majority of other methods for hormone analysis required animmunoassay. Immunoassays are expensive, specific to a particularanalyte and involve several steps.

[0032] (3) The present invention requires minimal sample preparationtime. For example, preparing a sample for hormone analysis can be donewithin 6 minutes.

[0033] (4) The procedure does not require a large sample size. A plasmaor serum sample can be as small as 100 μL for thyroid hormones. Thecurrent methods for hormone analysis that utilize mass spectrometryrequire 4-15 mL of plasma.

[0034] (5) The invention uses simple preparation techniques that areeasy to use and highly reproducible.

[0035] (6) The invention permits analysis to be performed on a varietyof sample types.

[0036] (7) The invention permits the analysis of hormones In a sample ofsaliva or urine which permits simple sample acquisition and the remotesubmission of samples to a clinic for analysis. In previous otherclinical methods, samples are taken by invasive means directly from thepatient in a clinic.

[0037] (8) The analysis by mass spectrometry is highly accurate. Inaddition, the procedure of the present invention is highly reproducible.

[0038] (9) The invention permits the analysis of a wide range of hormoneconcentrations. In addition, the limit of detection can be fairly low.

[0039] Accordingly, there is provided a use for a mass spectrometer forsimultaneously or sequentially analyzing a sample for a plurality ofhormones in a fast, simple and accurate way. The sample may be, forexample, serum, plasma, urine or saliva.

[0040] There is also provided a system for the fast, simple and accurateanalysis of a plurality of hormones comprising: reagents for thepreparation of the sample, reagents to perform the analysis on a massspectrometer, and a mass spectrometer to perform the analysis.

[0041] There is also provided a kit, comprising the various reagentsrequired for simultaneously or sequentially analyzing, within a sample,a plurality of hormones, including steroid hormones, thyroid hormonesand other hormones. The kit may include a standard solution of thehormones of interest, compounds as internal standards, mobile phasesolutions, methods and tools for separating hormones from samples, forexample HPLC columns, and quality control specimens.

[0042] There is also provided a method for the simultaneous orsequential analysis of one or more hormones comprising ionizing thehormones and analyzing the hormones by mass spectrometry.

[0043] Accordingly, there is also provided a method for the simultaneousor sequential analysis of one or more hormones comprising: obtaining asample containing or suspected of containing one or more hormones,removing proteins from the sample, separating the hormones from thesample, ionizing the hormones and analyzing the hormones in a massspectrometer.

[0044] Accordingly, there is also provided a method for the analysis ofone or more thyroid hormones comprising: obtaining a sample containingor suspected of containing one or more thyroid hormones, removingproteins from the sample, separating the thyroid hormone from thesample, ionizing the thyroid hormones, for example by electrosprayionization, and analyzing the hormone in a mass spectrometer, preferablyin the negative mode.

[0045] Accordingly, there is also provided a method for the simultaneousor sequential analysis of one or more thyroid hormones comprising,obtaining a sample containing or suspected of containing one or morethyroid hormones, removing proteins from the sample, separating thethyroid hormones from the sample, ionizing the thyroid hormone, forexample by electrospray ionization, and analyzing the hormones in a massspectrometer, preferably in the negative mode.

[0046] Accordingly, there is also provided a method for the simultaneousor sequential analysis of a plurality of thyroid hormones and aplurality of steroid hormones comprising: obtaining a sample containingor suspected of containing a plurality of hormones, removing proteinsfrom the sample, separating the hormones from the sample, ionizng thethyroid hormones, for example, by electrospray ionization, ionizing thesteroid hormones by photoionization, and analyzing the hormones in amass spectrometer, in the negative or positive modes.

BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The invention, including the best approaches known to theinventors, can be better understood with reference to the followingdetailed description taken in combination with the following drawings,in which:

[0048]FIG. 1 is a mass spectrum of a sample of plasma containing T4 andT3.

[0049]FIG. 2 is a mass spectrum of a globulin standard containing T4 andT3.

[0050]FIG. 3 is a typical tandem mass spectrometric chromatogramobtained for T4 and T3.

[0051]FIG. 4 is a graph showing T3 measured by Isotope Dilution TandemMass Spectrometry vs. Immunoassay.

[0052]FIG. 5 is a graph showing T4 measured by Isotope Dilution TandemMass Spectrometry vs. Immunoassay.

DETAILED DESCRIPTION OF THE EXEMPLIFIED EMBODIMENT

[0053] The invention provides methods of analysis for hormones. Thehormones may include:

[0054] Dehydroepiandrosterone (DHEA)

[0055] Dehydroepiandrosterone sulphate (DHEAS)

[0056] Aldosterone

[0057] Cortisol

[0058] 11-Deoxycorisol

[0059] Androstenedione

[0060] Testosterone

[0061] Estradiol

[0062] 17-OH Progesterone

[0063] Progesterone

[0064] Allopregnanolone

[0065] 16-OH Estrone

[0066] 2-OH Estrone

[0067] Estrone

[0068] Estriol

[0069] Vitamin D

[0070] thyroxine

[0071] free thyroxine

[0072] triiodothyronine

[0073] catecholamines

[0074] metanephrines

[0075] other steroid hormones

[0076] other thyroid hormones

[0077] other small peptide hormones

[0078] other amines

[0079] Sample

[0080] Any sample containing or suspected of containing a hormone can beused, including a sample of blood, plasma, serum, urine or saliva. Thesample may contain both free and conjugated or bound hormones. A samplesize of at least about 100 μL for hormones generally, or at least about700 μL for steroid hormones, is presently preferred.

[0081] Deproteinization

[0082] The sample is de-proteinated. This can be done by conventionaltechniques known to those skilled in the art. For example, a sample canbe de-proteinated with acetonitrile, containing internal standard,followed by vortexing and centrifugation. The internal standard may be,for example, the deuterated hormone.

[0083] Separation of Hormones from the Sample

[0084] The hormones are separated by methods known to those skilled inthe art. For example, the hormones may be separated by liquidchromatography through a column. The column may be a C-18 column. Thehormones are subsequently eluted from the column.

[0085] Introduction of Hormones into a Mass Spectrometer

[0086] The hormones are then introduced into a mass spectrometer.Optionally, the separation step and step of introducing the hormonesinto a mass spectrometer can be combined using a combined liquidchromatography spectrometry apparatus (LC/MS). This procedure is basedon an online extraction of the injected sample with subsequentintroduction into the mass spectrometer using a built-in switchingvalve.

[0087] Isotope Dilution Tandem Mass Spectrometry

[0088] Isotope dilution tandem mass spectrometry incorporates additionaldilution steps that act as an internal calibration so that anindependent isotopic reference material is not required. It avoids theneed to measure the isotope ratio of the highly enriched spike directly,and enables the final results to be arranged as a combination ofmeasurements that are largely insensitive to instrumental bias anddrift. Consequently, it has the potential to extend the scope ofapplication of isotope dilution tandem mass spectrometry to includeanalysis for which reference materials with certified isotope ratios arenot available or where contamination of the instrument by thehighly-enriched spike causes difficulty.

[0089] Instrumentation and Ionization Techniques

[0090] The hormones are subjected to ionization. Various ionizationtechniques can be used. For example, photoionization, electrosprayionization (ESI), atmospheric pressure chemical ionization (APCI), andelectron capture ionization may be used. Preferably, electrosprayionization is utilized when analyzing thyroid hormones.

[0091] The following mass spectrometers can be used: any tandem-massspectrometer, including hybrid quadrupole-linear ion trap massspectrometers and liquid chromatography-tandem mass spectrometers suchas the API 2000™ mass spectrometer the API 3000™ mass spectrometer, andthe API 4000™ mass spectrometer, described in U.S. Pat. Nos. 4,121,099;4,137,750; 4,328,420; 4,963,736; 5,179,278; 5,248,875; 5,412,208; and5,847,386 (Applied Biosystems/MDS SCIEX, Foster City, Calif./ConcordOntario, Canada). When analyzing thyroid hormones, a spectrometer with aturbo spray ion source, such as the API 2000™ and API 3000™ massspectrometers, is presently preferred. When analyzing FT4, the API 4000™mass spectrometer is presently preferred.

[0092] Ionization may be performed by utilizing the mass spectrometer inthe negative or the positive mode, depending on a particular analyte'stendency to give rise to a particular ion form, as is known to thoseskilled in the art. Typically, for thyroid hormones, the spectrometer isemployed in the negative mode.

[0093] Hormones are identified on the basis of the mass to charge ratioof their molecular ions and fragment ions, as is known to those skilledin the art. When the hormones are purified by liquid chromatography,they can also be identified by their retention times.

[0094] Hormones are quantified by their intensity as determined in themass spectrometer in counts per second. Calibration curves for knownconcentrations of the hormones are established for comparison.

EXAMPLES

[0095] The invention will now be demonstrated using the followingexamples, provided to demonstrate but not limit the embodiments of thepresent invention:

[0096] 1. Analysis of a sample for thyroid hormones

[0097] A sample of 100 μL of plasma was used. Proteins were precipitatedwith 150 μL of acetonitrile, capped and vortexed. The sample was thencentrifuged, and 200 μL of the supernatant was injected onto a SupelcoLC-18-DB™ chromatographic column equipped with Supelco Discovery C18™guard column, coupled to a tandem mass spectrometer (LC/MS/MS). Thecolumn was washed with 20% methanol in 5 mM ammonium acetate for 3minutes. The valve was switched and the sample was eluted in 75% to 95%methanol. The total run time was 6 minutes. Slight adjustments to thevolumes, concentrations and times described can be made, as is known tothose skilled in the art.

[0098] The eluant was introduced into an ion-spray ionization chamberand analyzed by API 2000™ mass spectrometer using the negative mode. Themass/charge ratios for T4 and T3 ions is 775.8 and 650 respectively. Theionization may be by electrospray using a turboionspray chamber.

[0099] This demonstrates a simple method of preparing a complexbiological matrix for analysis of hormone content, and a sensitiveanalytical method that permits the simultaneous analysis of twohormones, T3 and T4.

[0100] 2. Analysis of thyroid hormones using a methanol gradient toelute the hormones

[0101] A sample of 100 μL of plasma was used. Proteins were precipitatedwith 150 μL of acetonitrile, containing an internal standard ofdeuterated T₄, and vortexed. The sample was centrifuged, and 200 μL ofthe supernatant was injected onto a C-18 column coupled to a tandem massspectrometer (LC/MS/MS). The column was washed with 20% methanol in 5 mMammonium acetate for 3 minutes. The valve on the column was switched andthe sample was eluted in a methanol gradient of 20 to 100%. The totalrun time was 7 minutes. Slight adjustments to the volumes,concentrations and times described can be made by those skilled in theart.

[0102] A sample of the eluant was introduced into an ion-sprayionization chamber and analyzed by an AP 2000™ mass spectrometer usingthe negative mode. The ionization may be by electrospray using aturboionspray chamber. See FIG. 1 and FIG. 2 for mass spectrumsgenerated for T3 and T4.

[0103] This demonstrates a simple method of preparing a complexbiological matrix for analysis of thyroid hormone content, and asensitive analytical method that permits the simultaneous analysis ofmultiple hormones.

[0104] 3. Analysis of thyroid hormones using isotope dilution tandemmass spectrometry

[0105] This example describes an isotope dilution tandem massspectrometry method for the simultaneous determination of T4 and T3 inserum. The method is accurate, specific, precise (% CVs between 3.5 and9.0), simple—requiring no extraction and only protein precipitation andfast (<7 min).

[0106] Chemicals and Reagents

[0107] Standards of T4 and T3 were purchased from Sigma (St Louis, Mo.,USA). A stable deuterium-labeled internal standard, L-thyroxin-d₂ wassynthesized according to procedures described in the literature [16].[17] by Dr Tomas Class from the Chemistry Department at GeorgetownUniversity. HPLC grade methanol was purchased from VWR Scientific. Allother chemicals were of analytical grade and purchased from Sigma.

[0108] Solutions and Standards

[0109] Stock solutions of T3, T4 and internal standard (IS) wereprepared separately to obtain concentration of 1 mg/mL for each. 40%ammonium hydroxide (v/v) in methanol was used as a solvent. The analytestock solutions were diluted with methanol to obtain the spikingsolutions. The solutions were stored at 4° C. and could be used forseveral months. Standards for the calibration curve in the range of0.325 to 5 ng/mL for T3 and 12.5 to 200 ng/mL for T4 were prepared byadding the analyses to 3% human γ-globulin (volume of spiking solution<2% of final volume). Quality control (QC) samples (Diagnostic ProductCorp., Los Angeles, USA) at low, medium and high levels were used. Asolution of 50-ng/mL d₂-T4 in methanol was used as the internalstandard.

[0110] Sample Preparation

[0111] Serum/plasma samples were thawed at room temperature. 150 μL ofIS solution was added to aliquots of 100 μL of the serum or plasmasample. After 30 seconds of vortex mixing, the samples were stored for10 min at room temperature to allow complete protein precipitation. Thesamples were centrifuged for 10 min at 15,000 rpm and 100 μl ofsupernatant was injected into the LC-MS-MS system.

[0112] LC/MS/MS Conditions

[0113] An API 3000™ tandem mass-spectrometer (SCIEX, Toronto, Canada)equipped with turboionspray and Shimadzu HPLC system was used to performthe analysis. Negative ion multiple reaction-monitoring (MRM) mode wasused. The transitions to monitor were selected at m/z 650→127 for T3,m/z 776→127 for T4, m/z 778→127 for d₂-T4. Nitrogen served as auxiliary,curtain and collision gas. Gas flow rates, source temperature, ion Sprayvoltages and collision energies were optimized for every compound byinfusion of 1 μg/mL of the standard solutions in methanol at 20 μL/minand by flow-injection analysis (FIA) at LC flow rate. The main workingparameters for the mass spectrometer are summarized in Table 3. Dataprocessing was performed on Analyst 1.2 software package.

[0114] LC-MS-MS Procedure

[0115] The procedure is based on an online extraction/cleaning of theinjected samples with subsequent introduction into the mass-spectrometerby using a built-in Valco switching valve. 100 μl of the sample wereinjected onto a Supelco LC-18DB (3.3 cm×3.0 mm, 3.0 μm ID)chromatographic column equipped with a Supelco Discovery C-18 (3.0 mm)Guard column, where it underwent cleaning with 20% (v/v) methanol in 5mM ammonium acetate pH=4.0 at flow rate 0.8 mL/min. After 3.5 min ofcleaning the switching valve was activated, the column was flushed withwater/methanol gradient at flow rate 0.5 mL/min and the samples wereintroduced into the mass-spectrometer. The gradient parameters are shownin Table 4.

[0116] Immunoassays for T4 and T3

[0117] T4 was measured by the Dade RxL Dimension (Dade-BehringDiagnostics, Glasgow, Del.) and T3 by the DPC Immulite (DiagnosticProduct Corporation, Los Angeles, Calif.) according to themanufacturer's specifications.

[0118] Results

[0119] The optimal mass spectrometer working parameters are shown inTables 3 and 4.

[0120] Replicate sera were assayed both within-day and between-day atseveral concentrations. The within-day and between-day precision data isprovided in Tables 5 and 6.

[0121] Recovery studies for T4 and T3 are shown in Tables 7 and 8. Allresults shown are the means of 8 replicates.

[0122]FIG. 3 shows a typical tandem mass spectrometric chromatogramobtained for T3 and T4.

[0123] Specimens were tested for T3 and T4 by both immunoassay (T3 DPCImmulite, T4 Dade Behring Dimension RxL) and by tandem massspectrometry. Linear regression correlations (Prism) are shown in FIGS.4 and 5.

[0124] The lower limit of quantfication of the mass spectrometry methodwas found to be 0.15 ng/mL for both T3 and T4. Detection limit wasaround 0.062 ng/mL.

[0125] Discussion

[0126] Evidence initially gleaned from both the CAP PT Program andpediatric reference ranges employing different immunoassays indicatedthe probability of lack of specificity for T4 and T3 immunoassay tests.To adequately assess this phenomenon we developed the isotope dilutiontandem mass spectrometric method described in this example. Serum T4 andT3 detection methods have evolved through a variety of technologiessince the 1950s. Radioimmunoassay (RIA) methods to detect THs weredeveloped in the 1970s. Serum T4 and T3 concentrations are currentlymeasured by competitive immunoassay methods (IAs) that are mostlynon-isotopic and use enzymes, fluorescence or chemiluminescencemolecules as signals [27]. Table 2 clearly Indicates that current IAsfor T4 and T3 lack specificity and give mean results differing by afactor of approximately 2 in CAP PT programs. Total hormone assaysnecessitate the inclusion of a displacing agent (such as salicylate) torelease the hormone from its binding proteins [28]. The displacement ofhormone binding from serum proteins by such agents, together with thelarge sample dilution employed in modern assays, facilitates the bindingof hormone to the antibody reagent.

[0127] Since T3 is ten-fold lower in concentration compared with T4 inblood it therefore presents both a technical sensitive and precisionchallenge despite the use of a higher specimen volume. Although areliable high-range T3 measurement is critical for diagnosinghyperthyroidism, a reliable normal-range measurement is also importantfor adjusting antithyroid drug dosage and detecting hyperthyroidism insick hospitalized patients, in whom a paradoxically normal T3 value mayindicate hyperthyroidism.

[0128] The correlation coefficient for the T4 comparisons (0.931) issignificantly better than for the T3 comparisons (0.084) (FIGS. 4 and5). T3 by tandem mass spectrometry gave slightly higher results thanthose obtained by the DPC Immulite (FIG. 4). While this is true forchildren, our preliminary data for non-pregnant and pregnant womenindicates a very poor correlation for T3 in both groups (r between0.407-0.574).

[0129] The reasons for this are not clear but could includestandardization issues, heterophilic antibodies etc. Of importance, wedetermined that reverse T3, which lacks a daughter ion of 127 m/z,therefore does not interfere in our tandem mass spectrometry method.Applying the tandem mass spectrometric method to CAP PT samples in theK/KN general ligand program again revealed that around 85% of theimmunoassay methods gave means on samples which were lower than themeans obtained by our tandem mass spectrometry method while 15% hadhigher means.

[0130] In conclusion, correlations between immunoassays and tandem massspectrometry for T4 proved to be adequate except for the pregnantpopulation, while the data for T3 was far less impressive especiallyduring pregnancy. Recovery studies from several different sera usingdeuterated T4 as internal standard showed consistent (90-109%)recoveries for both T4 and T3 (Tables 7 and 8). The recovery differencesfound between samples were surprisingly larger for T4 than for T3. Thisindicates a lack of need to use deuterated T3 as the T3 internalstandard. The isotope dilution tandem mass spectrometric method wedeveloped is rapid (<7 min), accurate (provides the true result as hasbeen assessed by recovery studies), specific (measures only the analyteit purports to measure), precise (low % CV) and easy to perform. TABLE 3Tandem mass-spectrometer working parameters Parameter Value Nebulizergas (NEB) 8 Curtain gas (CUR) 10 Collision gas (CAD) 6 Turbolon SprayHeater gas 7 L/min Turbolon Spray (IS) voltage 4500 V Entrance Potential(EP) 7.5 V Collision cell Exit Potential (CXP) 5 V Source temperature450° Dwell time 250 msec

[0131] TABLE 4 Gradient parameters Time (min) Methanol (%) 3.50 75 5.2576 5.50 100 7.00 End

[0132] TABLE 5 Within day precision (n = 10) CONTROL 1 CONTROL 2 MeanMean Analyte (ng/mL) SD CV (%) (ng/mL) SD CV (%) T3 1.04 0.014 1.36 2.440.077 3.19 T4 24.1 0.437 1.81 81.2 1.502 1.85

[0133] TABLE 6 Between day precision (n = 20, 1 run per day for 20 days)CONTROL 1 CONTROL 2 CONTROL 3 Mean Mean Mean Analyte (ng/mL) SD CV (%)(ng/mL) SD CV (%) (ng/mL) SD CV (%) T3 1.08 0.05 4.47 2.39 0.22 9.213.49 0.31 9.00 T4 24.4 1.39 5.69 76.6 3.11 4.06 116.3 4.15 3.57

[0134] TABLE 7 Recovery of added thyroxine (T4) Added Detected Addedamount Sample # (ng/mL) mean recovered Recovery, % 1 (n = 8) 0 85.9 NA*NA 10 96.7 10.8 108.0 40 127.5 41.6 104.0 2 (n = 5) 0 62.6 NA NA 10 72.19.5 95.0 40 98.0 35.4 90.0 3 (n = 5) 0 73.8 NA NA 10 84.7 10.9 109.0 40116 42.2 105.0 4 (n = 5) 0 58.3 NA NA 10 68.0 9.7 97.0 40 95.0 36.7 92.0

[0135] TABLE 8 Recovery of added triiodothyronine (T3) Added DetectedAdded amount Sample # (ng/mL) mean recovered Recovery, % 1 (n = 8) 01.88 NA NA 0.25 2.12 0.24 96.0 1.00 2.85 0.97 97.0 2 (n = 5) 0 1.70 NANA 0.25 1.96 0.26 104.0 1.00 2.76 1.06 106.0 3 (n = 5) 0 1.56 NA NA 0.251.81 0.25 100.0 1.00 2.62 1.06 106.0 4 (n = 5) 0 0.49 NA NA 0.25 0.740.25 100.0 1.00 1.50 1.01 101.0

[0136] 4. Analysis of thyroid hormones and steroid hormones

[0137] A sample of 100 μL of plasma is used. Proteins are precipitatedwith 150 μL of acetonitrile and vortexed. The sample is centrifuged, and200 μL of the supernatant is injected onto a C-18 column coupled to atandem mass spectrometer (LC/MS/MS). The column is washed with 20%methanol in 5 mM ammonium acetate for 3 minutes. The valve on the columnis switched and the sample is eluted in a methanol gradient of 20 to100%. The total run time is 10 minutes. Slight adjustments to thevolumes, concentrations and times described can be made, as is known tothose skilled in the art.

[0138] A sample of the eluant is introduced into an ion-spray ionizationchamber and analyzed by API 3000™ mass spectrometer using the negativemode for thyroid hormones in the sample. Steroid hormones in the sampleare ionized by photoionization, with the spectrometer in the negative orpositive mode. Analysis in the positive mode is typically made for DHEA,Aldosterone, Cortisol, 11-Deoxycortisol, Androstenedione, Testosterone,Estradiol, 17-OH Progesterone, Progesterone, Allopregnalone, and VitaminD, whereas analysis in the negative mode is typically made for 16-OHEstrone, 2-OH Estrone, Estriol and DHEAS. However, it is possible toanalyze any of the hormones In either positive or negative mode.

[0139] This demonstrates a simple method of preparing a complexbiological matrix for analysis of possible steroid and thyroid hormonecontent, and a sensitive analytical method that permits the simultaneousanalysis of steroid and thyroid hormones.

[0140] The results indicate that this technique, allows for theidentification and characterization of low levels of thyroid hormone inhuman plasma and saliva.

[0141] While the above detailed description describes the exemplifyingembodiments of the present invention, it should be understood that thepresent invention is susceptible to modifications, variations andalterations without deviating from the scope of the invention.

REFERENCES

[0142] All references listed herein are incorporated by reference intheir entirety.

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What is claimed is:
 1. A method for mass spectrometric analysis of asample possibly containing one or more thyroid hormones, comprising thesteps: (a) providing a sample containing one or more thyroid hormones;(b) deproteinating the sample; (c) separating the one or more thyroidhormones from the sample; and (d) analyzing the one or more thyroidhormones using a mass spectrometer.
 2. The method according to claim 1wherein the one or more thyroid hormones are selected from the groupconsisting of T3, T4 and FT4.
 3. The method according to claim 1 whereinthe sample possibly containing one or more thyroid hormones is obtainedfrom a biological sample selected from the group consisting of blood,plasma, serum, urine and saliva.
 4. The method of claim 3 wherein thebiological sample is blood.
 5. The method of claim 3 wherein thebiological sample is plasma.
 6. The method of claim 3 wherein thebiological sample is serum.
 7. The method of claim 3 wherein thebiological sample is urine.
 8. The method of claim 3 wherein thebiological sample is saliva.
 9. The method according to claim 1 whereinsize of said sample containing one or more thyroid hormones Is at leastabout 100 μL.
 10. The method according to claim 1 wherein said step ofdeproteinating the sample comprises: (a) adding acetonitrile, containinginternal standards; (b) vortexing the sample; and (c) subjecting thesample to centrifugation.
 11. The method according to claim 1 whereinsaid step of deproteinating the sample comprises subjecting the sampleto precipitation with an agent containing internal standards, said agentselected from the group consisting of methanol, ethanol and salt. 12.The method according to claim 1 wherein said step of separating the oneor more thyroid hormones from the sample comprises introducing thesample to a liquid chromatography apparatus and subsequently eluting thethyroid hormones from the column.
 13. The method according to claim 12wherein said step of separating the one or more thyroid hormones fromthe sample comprises the use of a C-18 column.
 14. The method accordingto claim 1 wherein said step of separating the one or more thyroidhormones from the sample comprises the use of a combined liquidchromatography spectrometry apparatus.
 15. The method according to claim14 wherein the one or more thyroid hormones are introduced into a massspectrometer directly after being separated from the sample by way of anon-line extraction and use of a built-in switch valve.
 16. The methodaccording to claim 1 wherein the mass spectrometer is a liquidchromatography-tandem-mass spectrometer.
 17. The method according toclaim 16 wherein the liquid chromatography-tandem mass spectrometer isequipped with an electrospray ionization source.
 18. The methodaccording to claim 1 wherein said step of analyzing the one or morethyroid hormones using a mass spectrometer comprises an ionizationtechnique selected from the group consisting of photoionization,electrospray ionization, atmospheric pressure chemical ionization, andelectron capture ionization.
 19. The method according to claim 18wherein said ioniation technique is electrospray ionization.
 20. Themethod according to claim 18 wherein said ionization is performed inpositive mode.
 21. The method according to claim 18 wherein saidionization is performed in negative mode.
 22. The method according toclaim 1 wherein said step of analyzing the one or more thyroid hormonesusing a mass spectrometer comprises multiple reaction monitoring. 23.The method according to claim 1 wherein said step of analyzing the oneor more thyroid hormones using a mass spectrometer comprises selectedion monitoring.
 24. The method according to claim 1 wherein the samplecomprises a plurality of thyroid hormones and they are analyzedsimultaneously.
 25. The method according to claim 1 wherein the samplecomprises a plurality of thyroid hormones and they are analyzedsequentially.
 26. The method according to claim 1 wherein the sample isanalyzed by isotope dilution tandem mass spectrometry.
 27. A method ofinstructing an analysis of a sample that possibly contains one or morethyroid hormones, the method comprising providing instructions toprepare the sample according to steps (b) and (c) of claim 1 and analyzethe one or more thyroid hormones from the sample according to step (d)of claim
 1. 28. A method for mass spectrometric analysis of a samplepossibly containing one or more thyroid hormones and one or more steroidhormones, comprising the steps: (a) providing a sample containing one ormore thyroid hormones and one or more steroid hormones; (b)deproteinating the sample; (c) separating the one or more thyroidhormones and the one or more steroid hormones from the sample; and (d)analyzing the one or more thyroid hormones and the one or more steroidhormones using a mass spectrometer.
 29. The method according to claim 28wherein the thyroid hormones are selected from the group consisting ofT3, T4 and FT4.
 30. The method according to claim 28 wherein the steroidhormones are selected from the group consisting ofDehydroepiandrosterone (DHEA), Dehydroepiandrosterone sulphate (DHEAS),Aldosterone, Cortisol, 11-Deoxycortisol, Androstenedione, Testosterone,Estradiol, 17-OH Progesterone, Progesterone, Allopregnanolone, 16-OHEstrone, 2-OH Estrone, Estrone, and Estriol.
 31. The method according toclaim 28 wherein the sample is obtained from a biological sampleselected from the group consisting of blood, plasma, serum, urine andsaliva.
 32. The method of claim 31 wherein the biological sample isblood.
 33. The method of claim 31 wherein the biological sample isplasma.
 34. The method of claim 31 wherein the biological sample isserum.
 35. The method of claim 31 wherein the biological sample isurine.
 36. The method of claim 31 wherein the biological sample issaliva.
 37. The method according to claim 28 wherein size of said samplecontaining one or more thyroid hormones is at least about 100 μL. 38.The method according to claim 28 wherein size of said sample containingone or more steroid hormones is at least about 700 μL.
 39. The methodaccording to claim 28 wherein said step of deproteinating the samplecomprises: (a) adding acetonitrile, containing internal standards; (b)vortexing the sample; and (c) subjecting the sample to centrifugation.40. The method according to claim 28 wherein said step of deproteinatingthe sample comprises subjecting the sample to precipitation with anagent containing internal standards, said agent selected from the groupconsisting of methanol, ethanol and salt.
 41. The method according toclaim 28 wherein said step of separating the one or more thyroidhormones and the one or more steroid hormones from the sample comprisesintroducing the sample to a liquid chromatography apparatus andsubsequently eluting the hormones from the column.
 42. The methodaccording to claim 41 wherein said step of separating the one or morethyroid hormones and the one or more steroid hormones from the samplecomprises the use of a C-18 column.
 43. The method according to claim 28wherein said step of separating the one or more thyroid hormones and theone or more steroid hormones from the sample comprises the use of acombined liquid chromatography spectrometry apparatus.
 44. The methodaccording to claim 43 wherein the one or more thyroid hormones and theone or more steroid hormones are introduced into the mass spectrometerdirectly after being separated from the sample by way of an on-lineextraction and use of a built-in switch valve.
 45. The method accordingto claim 28 wherein the mass spectrometer is a liquidchromatography-tandem-mass spectrometer.
 46. The method according toclaim 45 wherein the liquid chromatography-tandem mass spectrometer isequipped with an atmospheric pressure ionization source.
 47. The methodaccording to claim 45 wherein the liquid chromatography-tandem massspectrometer is equipped with an electrospray ionization source.
 48. Themethod according to claim 28 wherein said step of analyzing the one ormore thyroid hormones and the one or more steroid hormones using a massspectrometer comprises an ionization technique selected from the groupconsisting of photoionization, electrospray ionization, atmosphericpressure chemical ionization, and electron capture ionization.
 49. Themethod according to claim 48 wherein said ionization is performed inpositive mode.
 50. The method according to claim 48 wherein saidionization is performed in negative mode.
 51. The method according toclaim 48 wherein said ionization is performed in positive mode ornegative mode.
 52. The method according to claim 28 wherein said step ofanalyzing the one or more thyroid hormones and the one or more steroidhormones using a mass spectrometer comprises multiple reactionmonitoring.
 53. The method according to claim 28 wherein said step ofanalyzing the one or more thyroid hormones and the one or more steroidhormones using a mass spectrometer comprises selected ion monitoring.54. The method according to claim 28 wherein the sample comprises aplurality of thyroid hormones and a plurality of steroid hormones, andthey are analyzed simultaneously.
 55. The method according to claim 28wherein the sample comprises a plurality of thyroid hormones and aplurality of steroid hormones, and they are analyzed sequentially. 56.The method according to claim 28 wherein the sample is analyzed byisotope dilution tandem mass spectrometry.
 57. A method of instructingan analysis of a sample that possibly contains one or more thyroidhormones and one or more steroid hormones, the method comprisingproviding instructions to prepare the sample according to steps (b) and(c) of claim 28 and analyze the one or more thyroid hormones and the oneor more steroid hormones from the sample according to step (d) of claim28.
 58. A system for the mass spectrometric analysis of a samplepossibly containing one or more thyroid hormones, comprising: (a)reagents for deproteinating the sample, including internal standards;(b) reagents for analyzing the one or more thyroid hormones using a massspectrometer; and (c) a mass spectrometer.
 59. The system according toclaim 58 wherein the mass spectrometer is a liquid chromatography-tandemmass spectrometer.
 60. A kit for use in mass spectrometric analysis of asample possibly containing one or more thyroid hormones, comprising: (a)reagents for deproteinating the sample, including internal standards;(b) reagents For separating the one or more thyroid hormones from thesample; (c) reagents for analyzing the one or more thyroid hormonesusing a mass spectrometer; (d) a solution of one or more thyroidhormones; and (e) instructions for analyzing the one or more thyroidhormones using a mass spectrometer.
 61. The kit according to claim 60further comprising: (a) mobile phase solutions; (b) a chromatographycolumn; and (c) a quality control specimen.
 62. Use of a massspectrometer for sequentially or simultaneously analyzing a samplepossibly containing a plurality of thyroid hormones.
 63. The useaccording to claim 62 wherein the mass spectrometer is a liquidchromatography-tandem mass spectrometer.